CN101650331B - Enzyme bioelectrochemical sensing chip and preparation method thereof - Google Patents
Enzyme bioelectrochemical sensing chip and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an enzyme bioelectrochemical sensing chip and a preparation method thereof, belonging to the technical fields of analysis and detection. In the invention, the screen printing technique which is suitable for large-scale batch production is used for preparing a conducting layer which is attached to a substrate insulating material and has a micro-nano hole structure, and the superimposed molecular adsorption layer technique is used for trapping high molecular dielectrics and biological enzymes in the micro-nano hole to prepare a working electrode of a biological sensing chip for quantitatively delectating solute molecules (such as glucose, cholesterol, blood ketone or lactic acid) in liquid samples (such as blood), and the sensing chip is especially suitable for the quantitative detection by the continuous method. The high molecular dielectrics and the biological enzymes in the biological sensing chip of the invention are in contact with each other in a molecular dispersed state and are tightly close to conducting particles, thus the quick electron transfer can be achieved to be favorable for improving the detection sensitivity of the sensing chip; and the superimposed molecular adsorption layer technique is used for trapping the high molecular dielectrics and the biological enzymes, thus the semi-dialysis membrane has no need of being used singly, and the intercepting effect is better.
Description
Technical field
The present invention relates to a kind of enzyme bioelectrochemical sensing chip that is used for the specific solutes content of tracer liquid, also relate to the preparation method of this bio-sensing chip simultaneously, belong to technical field of analysis and detection, comprise medical treatment, environmental protection, food security or the like.
Background technology
Characteristics such as the enzyme bioelectrochemical sensing device is a kind of novel analysis and detecting instrument equipment, has the degree of accuracy height, and selectivity is strong, and speed is fast, and volume is little, and cost is low, and is simple to operate and easy-to-use.Therefore it all has huge applications potentiality and development prospect in various fields, for example health care, environmental protection and food security etc.Enzyme bioelectrochemical sensing chip (abbreviation bio-sensing chip) is the core component of enzyme bioelectrochemical sensing device.It mainly is made up of molecule receiving element (receptor) and two parts of signal sensing element (transducer).The former normally a kind of biology enzyme causes the change of molecule chemical property to the catalytic action that detects the solute redox reaction through biology enzyme.Signal sensing element converts the change of this chemical property to measurable electric signal (like electric current or electric capacity) again.Because the particular organisms enzyme is to the strong selectivity of target molecule, thereby can avoid the interference of other complicated composition in the test liquid, realize accurate quantitative analysis.Signal sensing element generally comprises dielectric (mediator) and conductive material etc.Usually bio-sensing chip also can have independently contrast electrode (reference electrode) composition sometimes by a working electrode (working electrode) and a counter electrode (counter electrode).
Aspect the development and application of enzyme bioelectrochemical sensing device, the blood-sugar detecting instrument (SMBG) that is used for the blood-glucose detection has been obtained great success, and has many patents to deliver.Its product can help the patient to carry out self-blood sugar test, thereby for daily state of illness monitoring, control and the treatment of diabetes provides help, has obtained remarkable economical and social benefit.Yet the blood sugar test bio-sensing chip of being developed so far (being commonly called as " detection strip ") can only disposablely use, thereby has determined the limitation of this series products.For example; This " single-point type " measured and can not be realized blood sugar level real " in good time " is grasped; Thereby when the blood sugar level fluctuation is hanged down than big and/or test frequency, omit probably " key point ", and do not reach the purpose that effectively prevents hypoglycemia or hyperglycaemia.For this reason, the scientific worker is making great efforts to develop the continous way blood sugar test product that has the bio-sensing chip that can use continuously in recent years.Its major advantage comprises:
At any time grasp blood sugar level and fluctuation situation; And (like the too fast or approaching critical value of setting of blood sugar level change) pointed out patient or medical personnel automatically where necessary; Prevent the generation of hypoglycemia or hyperglycaemia to take measures, successfully realize effective control of blood glucose fluctuation scope.In addition, also be particularly suitable for the inpatient, children and old man, and the monitoring of particular case (like driving and sleep etc.).
Detail record different time sections (as night, every day reaching and wait weekly) blood sugar level and fluctuation data (can download), the state of an illness of grasping the patient for the doctor provides reliable basis with the formulation medical scheme.
Can with the insulin pump logotype, for the patient provides " artificial pancreas " (artificial pancreas).
The diagnosis and the monitoring equipment that can be used as hospital's blood sugar are like patient's (being not limited to the diabetic) operation and postoperative care etc.
Though continuity method bio-sensing chip and disposable biological sensing chip all are based on the principle of work of enzyme bioelectrochemical, the difference of both request for utilizations has determined the former must adopt new mentality of designing and preparation method.Key wherein be prevent effectively on the working electrode main redox reagent (like biology enzyme and dielectric etc.) in test process loss (leach out) and keep their activity.The method that is adopted at present mainly is divided into chemical bonding and physics is held back (entrapment) two kinds.The chemical bonding method generally is to adopt chemical reaction to form molecule crosslinked network with the redox reagent on the working electrode self or with other auxiliary reagents.This molecule crosslinked network is forming swelling and the undissolved hydrosol with tracer liquid (like a blood or a plastid liquid (interstitial-fluid)) when contacting.Because redox reagent has become the part of this molecule crosslinked network, so can not run off easily.The patent example of this method has US6299757 and US6461496, also has a large amount of scientific and technical literatures to deliver, for example list of references 5-7.But the chemical bonding method can cause the decline significantly of bioenzyme activity usually, if control improper even can cause the bio-sensing chip job insecurity.It is to adopt half dialysis membrane (membrane) to keep apart attached to the redox reagent and the tracer liquid on conductive material surface that typical physics is held back method.WO2005048834 and US7432069 are the patents that adopts this method.Here the control of half dialysis membrane performance is very crucial, comprises thickness of surface property, fenestra size and its distribution and film or the like.On the one hand, prevent or slow down the loss of redox reagent; On the other hand, other important performance of balance bio-sensing chip again, for example solute molecule diffuses through the speed of fenestra, the formation of solute concentration gradient and foundation of balance or the like.The manufacturing process of this type bio-sensing chip is complicated, difficulty is bigger, is unfavorable for scale of mass production.
The technology (layer-by-layer) of utilizing charge interaction in recent years and forming stack molecular adsorption layer has obtained increasing concern in the research and development field of bio-sensing chip, obtained some progress (referring to list of references 8-11).But research work so far mainly concentrates on the employing precious metal, and is for example golden, as conductive base.The preparation process need of this type of bio-sensing chip carries out complicated pretreatment to the metallic conduction substrate surface, and also very high to the preparation environment requirement.These have all increased the manufacture difficulty and the cost of bio-sensing chip.
List of references:
1, patent US6299757.
2, patent US6461496.
3, patent WO2005048834.
4, patent US7432069.
5、L?S?Bean，et?al，“The?electrochemical?behavior?of?ferrocene?in?aphotocurable?poly(methyl?methacrylate-co-2-hydroxyethyl?methacrylate)film?for?aglucose?biosensor”，Bioelectrochemistry，2005，65，pp157-162.
6、H-z?Bu，et?al，“Charaterization?of?a?ferrocene-containing?polyacrylamide-based?redox?gel?for?biosensor?use”，Analytical?Chemistry，1995，67，pp4071-4076.
7、E.J.Calvo?and?R.Etchenique，“electrical?communication?betweenelectrodes?and?enzymes?mediated?by?redox?hydrogels”，Analytical?Chemistry，1996，68，pp4186-4193.
8、H.C.Yoon?and?H-S.Kim，“Multilayered?assembly?of?dendrimers?withenzymes?on?gold：thickness-controlled?biosensing?interface”，Analytical?Chemistry，2000，72，pp922-926.
9、H.C.Yoon，et?al，“Functionalization?of?a?poly(amidoamine)dendrimer?withferrocenyls?and?its?application?to?the?construction?of?a?reagentless?enzyme?electrode”，Analytical?Chemistry，2000，72，pp4420-4427.
10、S.Zhang，et?al，“Multilayered?construction?of?glucose?oxidase?andpoly(allylamine)ferrocene?on?gold?electrodes?by?means?of?layer-by-layer?covalentattachment”，Sensor?and?Actuators?B，2004，101，pp387-393.
11、S.Suye?et?al，“Layer-by-layer?assembly?of?enzymes?and?polymerizedmediator?on?electrode?by?electrostatic?adsorption”，Science?and?Technology?ofAdvanced?Materials，2004，5，pp371-376.
12、Saito，Takahiro，et?al.，″Characterization?of?poly(vinylferrocene-co-2-hydroxyethyl?methacrylate)for?use?as?electron?mediator?in?enzymatic?glucose?sensor″，Reactive?&?Functional?Polymers，1998，37，pp263-269.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme bioelectrochemical sensing chip and preparation method thereof; Can be used as the working electrode that the solute molecule (for example glucose) in the liquor sample (for example blood) is carried out the bio-sensing chip of detection by quantitative to process, be specially adapted to the continuity method detection by quantitative.
Technical scheme of the present invention: a kind of enzyme bioelectrochemical sensing chip, its working electrode is made up of base insulating material (101), micro-nano porous conductive material layer (102) and conducting wire (103) and the macromolecule dielectric and the adsorbed layer of biology enzyme that are trapped among the micro-nano hole.
The preparation method of said enzyme bioelectrochemical sensing chip; The present invention is through reasonable material and proportioning; Its working electrode adopts the screen printing technique that is fit to scale of mass production successfully to prepare attached to the porous conductive material layer (102) with charged micro-nano pore space structure on the base insulating material (101) and conducting wire (103) (Fig. 1); In conjunction with stack molecular adsorption layer technology special synthetic charged macromolecule dielectric and biology enzyme are trapped among the micro-nano hole; Can be used as the working electrode that the solute molecule in the liquor sample (for example blood) (for example glucose, cholesterol, blood ketone or lactic acid) is carried out the enzyme bioelectrochemical sensing chip of detection by quantitative to process, be specially adapted to the continuity method detection by quantitative.
Working electrode of the present invention should with at least one counter electrode or contrast electrode, or counter electrode and a contrast electrode are shared to form bio-sensing chip.Counter electrode and contrast electrode can be with working electrode of the present invention attached to (Ag/AgCl that for example forms) on the same base insulating material through the serigraphy drying; Also can be attached on another base insulating material, can also be self-existent (for example platinum wire is as counter electrode etc.).
The preparation process of enzyme bioelectrochemical sensing chip working electrode is:
(1) preparation electrically conductive ink,
(2) prepare the base insulating material,
(3) serigraphy,
(4) dryness finalization,
(5) alternately absorption and firm of macromolecule dielectric and biology enzyme;
Said base insulating material is selected the plastic foil or the plastic sheet of polyester, polybutylene terephthalate, polystyrene, polycarbonate, polyurethane, PVC or their potpourri for use;
Said micro-nano porous conductive material layer is made up of conductive particulate materials and polymer binder;
Said macromolecule dielectric is selected for use directly the micromolecule dielectric is connected on the positively charged macromolecular chain through the reaction between the chemical functional group; Or through selecting for use two or more monomers to carry out copolymerization; Said biology enzyme is selected GDH or glucose oxidase for use; Cholesterol oxidase or cholesterin dehydrogenase; β-hydroxyl (base) butyryl dehydrogenase; Or lactic dehydrogenase.
Below describe with regard to principal ingredient used in the present invention:
(1) base insulating material
The sheet material or the film of all kinds of insulating material of widely using at present all can be used for the present invention.For example, various plastic foils and plastic sheet comprise polyester (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), polyurethane (PU), PVC (PVC) and their potpourri or the like.
(2) conductive particle
Any conductive particle with certain size size all can be used for the present invention.For example, carbon black particle (carbon black), graphite granule (graphite particle), gold particle, platinum particle (platinum), platinum and palladium (palladium) alloy particle, the carbon black particle of platinum modification or graphite granule of platinum modification or the like.The size of conductive particle can be 1 nanometer to 50 micron.The selection of conductive particle size should be considered the aperture of micro-nano hole; Guarantee that again formed porous, electrically conductive layer has excellent conducting performance.The general potpourri that preferably uses two kinds or the conductive particle that both are above.
(3) polymer binder (binder)
The polymer binder that the present invention selects for use has the function of two aspects: one of which is to process printing ink with above-mentioned conductive particle and other auxiliary reagent, forms complete conductive layer after the serigraphy drying; Its two, self has the particular functional group polymer binder, for example-N
+Or-COO
-, cause the hole wall of micro-nano hole to have electric charge under certain condition, so that realize macromolecule dielectric and the successful absorption of biology enzyme in micro-nano hole.
Synthetic and natural polymer binder all can be used for the present invention.Can use separately, also can be that two or more share.The polymer binder of synthetic can be a homopolymer, also can be binary or multiple copolymer.For example, vinyl class in polymer (vinyl polymer), propylene esters polymer (acrylate polymer), polyurethane polymer (polyurethane), epoxy-based polymer (epoxy resin), or the like.Natural polymer binder also can re-use after the modification.
Polymer binder can be water miscible, also can be dissolved in organic solvent; Can be linear, also can be the cross-linking type that crosslinked monomer or oligomer form in the preparation process of micro-nano porous conductive material layer, but after dry with no longer water-soluble and neutral aqueous solution.
(4) macromolecule dielectric
Mostly common dielectric is micromolecular compound, can not satisfy the requirement of this patent.Thereby be necessary to utilize macromolecule synthesising technology that different types of micromolecule dielectric is connected on the macromolecular chain through chemical bonded refractory.Simultaneously, also to consider during Polymer Synthesizing and introduce the functional group that has positive charge under the certain condition.Synthetic approach can be directly the micromolecule dielectric to be connected to (referring to the embodiment of the invention 1) on the positively charged macromolecular chain through the reaction between the chemical functional group; Also can be through selecting for use two or more monomers to carry out copolymerization (referring to the embodiment of the invention 3).
Starting material of being selected for use in the embodiment of the invention and synthetic method are merely the purpose of demonstration, can change as required in the practical operation, also can utilize the technology and/or the new method and adopt new route of synthesis of association area.
(4) biology enzyme
The present invention can select corresponding biology enzyme according to the request for utilization of detection needs and/or bio-sensing chip, and for example preparation is used for the bio-sensing chip of glucose detection, can select GDH or glucose oxidase for use.The former can overcome the oxysensible deficiency of the latter.Can also be according to the different choice and the corresponding biology enzyme of solute to be detected of solute to be detected, for example select for use cholesterol oxidase (cholesteroloxidase) or cholesterin dehydrogenase (cholesterol dehydrogenase) preparation be used for cholesterol detection bio-sensing chip, (β-hydroxybutyratedehydrogenase) preparation is used for the bio-sensing chip that blood ketone detects to select β-hydroxyl (base) butyryl dehydrogenase for use; Select the bio-sensing chip of lactic dehydrogenase (lactatedehydrogenase) preparation lactate detection for use, or the like.
(5) surface film
It mainly is the biocompatibility that improves bio-sensing chip for further that the present invention uses surface film, and then guarantees measuring accuracy and the accuracy in the use.Here surface film not necessarily will be born the function that physics is held back biology enzyme and macromolecule dielectric, so any membrane material with good biocompatibility all can be used for the present invention.For example can adopt prefabricated dialysis membrane or half dialysis membrane; Or the micro-nano porous conductive material laminar surface in-place forming of accomplishing (in-situ formation) that has macromolecule dielectric and biology enzyme.
Find in the experiment that for example prepared bio-sensing chip itself has excellent biological compatibility in the embodiment of the invention 3, so whether the use of surface film can be decided according to actual conditions.
Preparation electrically conductive ink: at first through forming the solution of polymer binder in the potpourri of polymer binder is water-soluble, organic solvent or water and organic solvent; Again conductive particulate materials is dispersed in the solution of polymer binder the slurry that the abundant shear-mixed of warp is easy to deposit with formation, i.e. electrically conductive ink.
Prepare the base insulating material: be attached with the conducting wire on the base insulating material in advance; And/or be attached with counter electrode in advance or/and contrast electrode.
Serigraphy and dryness finalization: adopt fast deposition process, high-precision silk screen printing is deposited on electrically conductive ink on the base insulating material; Utilize heat, light, microwave or their acting in conjunction that the volatile ingredient solvent in the electrically conductive ink is removed, form micro-nano porous conductive material layer.
Macromolecule dielectric and biology enzyme be absorption and firm alternately: positively charged macromolecule dielectric relies on charge interaction and is adsorbed on the hole wall of micro-nano porous conductive material layer, forms positively charged macromolecule dielectric adsorbed layer; Electronegative redox biology enzyme relies on charge interaction and is adsorbed on the formed positively charged macromolecule dielectric adsorbed layer, forms electronegative redox biology enzyme adsorbed layer; The absorption of macromolecule dielectric and biology enzyme alternately repeats absorption on demand, to form many adsorbed layers.
The application of described enzyme bioelectrochemical sensing chip; It is used for blood testing; Corresponding biology enzyme is selected in request for utilization according to detection needs and/or sensing chip, selects for use the preparation of GDH or glucose oxidase to be used for the bio-sensing chip of glucose detection; Select for use the preparation of cholesterol oxidase or cholesterin dehydrogenase be used for cholesterol detection bio-sensing chip, select for use β-hydroxyl (base) butyryl dehydrogenase preparation to be used for the bio-sensing chip that blood ketone detects; Select for use the lactic dehydrogenase preparation to be used for the bio-sensing chip of lactate detection.
The preparation flow synoptic diagram of bio-sensing chip working electrode of the present invention is seen Fig. 2.
Step among Fig. 2 can be changed according to concrete needs.For example, step 5-8 can repeat the total adsorbance with control macromolecule dielectric and biology enzyme.
Beneficial effect of the present invention: compare with aforesaid chemical crosslink technique; Macromolecule dielectric and biology enzyme in the bio-sensing chip of the present invention are in contact with one another with the molecule disperse state; And next-door neighbour's conducting particles, thereby can realize that swift electron shifts, and helps improving the detection sensitivity of sensing chip.In addition, micro-nano hole has electric charge, has very strong water wettability, in case sensing chip contact with liquid to be detected, hydrone just can get into rapidly and quick " preheating " that realize sensing chip (conditioning).Hold back method with aforesaid half dialysis membrane physics and compare, the present invention relies on stack molecular adsorption layer technology to combine with micro-nano pore space structure to holding back of macromolecule dielectric and biology enzyme and realizes, do not need to use half dialysis membrane separately, holds back better effects if.
Description of drawings
The synoptic diagram of Fig. 1 bio-sensing chip working electrode of the present invention.
The preparation flow synoptic diagram of Fig. 2 bio-sensing chip working electrode of the present invention.
Bio-sensing chip 1 kinetic current of Fig. 3 embodiment of the invention 1 preparation is with the variation of concentration of glucose in the phosphate buffer (PBS).
The kinetic current of glucose is to the calibration curve of concentration of glucose mapping in 2 pairs of blood of bio-sensing chip of Fig. 4 embodiment of the invention 3 preparations.
Bio-sensing chip 3 kinetic currents of Fig. 5 embodiment of the invention 5 preparations are with the variation of concentration of glucose in the phosphate buffer (PBS), and the kinetic current of glucose is to the calibration curve of concentration of glucose mapping.
Embodiment
Through embodiment the present invention is further specified below.
The preparation of embodiment 1 micro-nano multiporous biological sensing chip working electrode 1
The electrically conductive ink that present embodiment is used is a carbon printing ink.The prescription and the proportioning of carbon printing ink are listed in table 1.
Table 1
| Material | Percentage by weight |
| The 35%-45% WS of carboxylic linear macromolecule A (styrene of hydrolysis and maleic anhydride base co-polymer) | 45-65 |
| Carbon black granules | 10-15 |
| Graphite granule | 15-20 |
| Tween80 (Tween 80) spreading agent | 2-10 |
| Water | 0-10 |
Prescription and the prepared carbon printing ink of proportioning of pressing table 1 form the micro-nano porous, electrically conductive layer attached to polyester base insulation sheet surface through serigraphy and drying.Its thickness is the 5-50 micron.Baking temperature and time are respectively 55-125 ℃ and 5-30 minute.
The macromolecule dielectric is in methyl alcohol, (carbonyldiimidazole) to make catalyzer with carbonyl dimidazoles (CDI) by carboxylic acid ferrocene (vinylferrocene carboxylic acid) and polyethyleneimine (polyethyleneimine), 3-5 hour gained of reaction under room temperature.
Biology enzyme is a glucose oxidase.
The absorption of macromolecule dielectric and biology enzyme is that the micro-nano porous, electrically conductive layer attached to polyester base insulation sheet surface with above-mentioned preparation successively is immersed among the solution (1-5 mg/ml) of macromolecule dielectric and biology enzyme 5-20 minute respectively, and final drying forms.
The test of embodiment 2 micro-nano multiporous biological sensing chip working electrodes 1
Form bio-sensing chip with embodiment 1 prepared bio-sensing chip working electrode 1 and by the prepared Ag/AgCl contrast electrode of serigraphy.Its test to the glucose response electric current is to use an All-China Federation of Taiwan Compatriots that the steady potentiometer of computing machine is arranged; Keep 0.4 volt of stable potential; Two electrodes are fixed on one side by side to be had in the small-sized flow cell of import and export (flow cell); PBS (pH 7.4) with different concentration of glucose crosses two electrode surfaces with the flow velocity continuous stream of 10-15 cm per minute, and the continuous recording bio-sensing chip is to the variation of glucose and concentration thereof.Concentration of glucose regularly uses YSI (YSI Inc., YellowSprings blood sugar analyzer) to demarcate.
Fig. 3 shows test results: the sensing chip kinetic current is with the variation of concentration of glucose among the PBS.
The preparation of embodiment 3 micro-nano multiporous biological sensing chip working electrodes 2
Except materials used, the preparation process of micro-nano multiporous biological sensing chip working electrode 2 is identical with embodiment 1 with method.
The macromolecule dielectric here is to be formed through radical copolymerization by ethene ferrocene (vinylferrocene), the two ethylamine ethyl fat (2-(diethylammonia) ethyl methacrylate) of methacrylic acid-2-and methacrylic acid-2-hydroxyethyl fat (2-(hydroxyethyl) methacrylate).Details are referring to the list of references 12 of this patent.Biology enzyme is a GDH.
The test of embodiment 4 micro-nano multiporous biological sensing chip working electrodes 2
Form bio-sensing chip with embodiment 3 prepared working electrodes 2 and by the prepared Ag/AgCl contrast electrode of serigraphy.Its test to the glucose response electric current is to use an All-China Federation of Taiwan Compatriots that the steady potentiometer of computing machine is arranged; Keep 0.4 volt of stable potential; Two electrodes are fixed on one side by side to be had in the small-sized flow cell of import and export; The blood of different concentration of glucose was crossed two electrode surfaces about 8 hours with the flow velocity continuous stream of 10-15 cm per minute, and the continuous recording bio-sensing chip is to the variation of glucose in the blood and concentration thereof.Concentration of glucose is regularly demarcated with YSI in the blood.
Fig. 4 shows the calibration curve of sensing chip 2 kinetic currents to concentration of glucose mapping in the blood.
The preparation of embodiment 5 micro-nano multiporous biological sensing chip working electrodes 3
Except the employed polymer binder of preparation carbon printing ink, preparation material, process and the method for micro-nano multiporous biological sensing chip working electrode 3 are identical with embodiment 3.
Here polymer binder is the 35%-45% WS of carboxylic linear macromolecule B (styrene of partial hydrolysis and maleic anhydride base co-polymer).
Prepared working electrode 3 surfaces cover polysulfones (polysulfone) surface film of one deck moulding in advance again.
The test of embodiment 6 micro-nano multiporous biological sensing chip working electrodes 3
Test with the prepared 3 pairs of glucose response electric currents of bio-sensing chip working electrode of embodiment 5 is to use an All-China Federation of Taiwan Compatriots that the steady potentiometer (potentialstat) of computing machine is arranged; Ag/AgCl makes contrast electrode and platinum filament is made counter electrode; 0.4 volt of stable potential is carried out among the PBS of 5 milliliters pH 7.4.The change in concentration of glucose is to lean on
Substep adds 0.5 mole glucose solution or dilutes with phosphate buffer (PBS) to be realized.
Fig. 5 shows the experimental record of sensing chip 3 kinetic currents, and electric current is to the calibration curve of concentration of glucose mapping among the PBS.
Except that the foregoing description, the present invention can also have other embodiment method.All employings are equal to design that replacement or equivalent transformation form, prescription, proportioning, technical scheme or the like, are the protection domain of requirement of the present invention.
Claims (8)
1. the preparation method of an enzyme bioelectrochemical sensing chip; It is characterized in that its working electrode adopts the screen printing technique preparation that is fit to scale of mass production attached to having micro-nano porous conductive material layer (102) and conducting wire (103) on the base insulating material (101); In conjunction with stack molecular adsorption layer technology macromolecule dielectric and biology enzyme are trapped among the micro-nano hole, process the working electrode that the solute glucose in the blood sample, cholesterol, blood ketone or lactic acid molecules is carried out the enzyme bioelectrochemical sensing chip of detection by quantitative with work;
The preparation process of enzyme bioelectrochemical sensing chip working electrode is:
(1) preparation electrically conductive ink,
(2) prepare the base insulating material,
(3) serigraphy,
(4) dryness finalization,
(5) alternately absorption and firm of macromolecule dielectric and biology enzyme;
Said base insulating material is selected the plastic foil or the plastic sheet of polyester, polybutylene terephthalate, polystyrene, polycarbonate, polyurethane, PVC or their potpourri for use;
Said micro-nano porous conductive material layer is made up of conductive particulate materials and polymer binder;
Said macromolecule dielectric is selected for use directly the micromolecule dielectric is connected on the positively charged macromolecular chain through the reaction between the chemical functional group; Or through selecting for use two or more monomers to carry out copolymerization;
Said biology enzyme is selected GDH or glucose oxidase for use; Cholesterol oxidase or cholesterin dehydrogenase; β-hydroxyl (base) butyryl dehydrogenase; Or lactic dehydrogenase.
2. preparation method according to claim 1 is characterized in that the conductive particulate materials of being selected for use is two or more the potpourri in the following conductive particulate materials: the carbon black particle of carbon black particle, graphite granule, gold particle, platinum particle, platinum and palldium alloy particle, platinum modification or the graphite granule of platinum modification.
3. preparation method according to claim 1 is characterized in that the polymer binder of being selected for use has the function of two aspects: one of which is to process printing ink with above-mentioned conductive particulate materials and auxiliary reagent, forms complete conductive layer after the serigraphy drying; Its two, self has the particular functional group polymer binder :-N
+Or-COO
-, cause the hole cornice of micro-nano hole that electric charge is arranged, realize macromolecule dielectric and the absorption of biology enzyme in micro-nano hole;
Select synthetic or natural polymer binder for use; Use separately, two or more share; The polymer binder of synthetic is homopolymer, binary or multiple copolymer; Re-use after natural polymer binder or the modification;
Polymer binder is selected for use water miscible, or is dissolved in organic solvent; Be the polymkeric substance of line style, or the cross-linking type that in the preparation process of micro-nano porous conductive material layer, forms of crosslinked monomer or oligomer; But it is dry afterwards with no longer water-soluble or neutral aqueous solution.
4. preparation method according to claim 3 is characterized in that the polymer binder of synthetic is selected vinyl class in polymer, propylene esters polymer, polyurethane polymer or epoxy-based polymer for use.
5. preparation method according to claim 1 is characterized in that preparing electrically conductive ink: at first through forming the solution of polymer binder in the potpourri of polymer binder is water-soluble, organic solvent or water and organic solvent; Again conductive particulate materials is dispersed in the solution of polymer binder the slurry that the abundant shear-mixed of warp is easy to deposit with formation, i.e. electrically conductive ink.
6. preparation method according to claim 1 is characterized in that preparing the base insulating material: be attached with the conducting wire on the base insulating material in advance; And/or be attached with counter electrode in advance or/and contrast electrode.
7. preparation method according to claim 1 is characterized in that serigraphy and dryness finalization: adopt fast deposition process, high-precision silk screen printing is deposited on electrically conductive ink on the base insulating material; Utilize heat, light, microwave or their acting in conjunction that the volatile ingredient solvent in the electrically conductive ink is removed, form micro-nano porous conductive material layer.
8. preparation method according to claim 1; It is characterized in that the alternately absorption and firm of macromolecule dielectric and biology enzyme: positively charged macromolecule dielectric relies on charge interaction and is adsorbed on the hole wall of micro-nano porous conductive material layer, forms positively charged macromolecule dielectric adsorbed layer; Electronegative redox biology enzyme relies on charge interaction and is adsorbed on the formed positively charged macromolecule dielectric adsorbed layer, forms electronegative redox biology enzyme adsorbed layer; The absorption of macromolecule dielectric and biology enzyme alternately repeats absorption on demand, to form many adsorbed layers.
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| JP6205545B2 (en) * | 2012-06-25 | 2017-10-04 | 合同会社バイオエンジニアリング研究所 | Enzyme electrode |
| CN107064262A (en) * | 2017-05-07 | 2017-08-18 | 莆田方家铺子食品营养研究中心 | Cholesterol Biosensor based on graphene/titanium dioxide/platinum palladium nano composite material and preparation method and application |
| TWI703323B (en) * | 2018-11-14 | 2020-09-01 | 及安生物科技股份有限公司 | Method for simultaneously detecting glucose concentration and percentage of glycated hemoglobin in single test strip |
| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
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| CN101163964A (en) * | 2005-10-17 | 2008-04-16 | 住友电气工业株式会社 | Biosensor chip and process for producing the same |
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| CN1460852A (en) * | 2003-06-18 | 2003-12-10 | 清华大学 | Multifunctional blood detection chip |
| CN1563969A (en) * | 2004-03-22 | 2005-01-12 | 南开大学 | Biological enzyme electrode for biosensor, and its prepn. method |
| CN101163964A (en) * | 2005-10-17 | 2008-04-16 | 住友电气工业株式会社 | Biosensor chip and process for producing the same |
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